IL11-mediated stromal cell activation may not be the master regulator of pro-fibrotic signaling downstream of TGFß.

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFß. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes. We confirmed increased mRNA expression of IL11 and IL11Rα in fibrotic diseases by OMICs approaches and in situ hybridization. However, the vital role of IL11 as a driver for fibrosis was not recapitulated. While induction of IL11 secretion was observed downstream of TGFß signaling in human lung fibroblasts and epithelial cells, the cellular responses induced by IL11 was quantitatively and qualitatively inferior to that of TGFß at the transcriptional and translational levels. IL11 blocking antibodies inhibited IL11Rα-proximal STAT3 activation but failed to block TGFß-induced profibrotic signals. In summary, our results challenge the concept of IL11 blockade as a strategy for providing transformative treatment for fibrosis.

AllergoOncology: Danger signals in allergology and oncology: A European Academy of Allergy and Clinical Immunology (EAACI) Position Paper.

The immune system interacts with many nominal 'danger' signals, endogenous danger-associated (DAMP), exogenous pathogen (PAMP) and allergen (AAMP)-associated molecular patterns. The immune context under which these are received can promote or prevent immune activating or inflammatory mechanisms and may orchestrate diverse immune responses in allergy and cancer. Each can act either by favouring a respective pathology or by supporting the immune response to confer protective effects, depending on acuity or chronicity. In this Position Paper under the collective term danger signals or DAMPs, PAMPs and AAMPs, we consider their diverse roles in allergy and cancer and the connection between these in AllergoOncology. We focus on their interactions with different immune cells of the innate and adaptive immune system and how these promote immune responses with juxtaposing clinical outcomes in allergy and cancer. While danger signals present potential targets to overcome inflammatory responses in allergy, these may be reconsidered in relation to a history of allergy, chronic inflammation and autoimmunity linked to the risk of developing cancer, and with regard to clinical responses to anti-cancer immune and targeted therapies. Cross-disciplinary insights in AllergoOncology derived from dissecting clinical phenotypes of common danger signal pathways may improve allergy and cancer clinical outcomes.

FcRn is a CD32a coreceptor that determines susceptibility to IgG immune complex-driven autoimmunity.

IgG immune complexes (ICs) promote autoimmunity through binding fragment crystallizable (Fc) γ-receptors (FcγRs). Of these, the highly prevalent FcγRIIa (CD32a) histidine (H)-131 variant (CD32aH) is strongly linked to human autoimmune diseases through unclear mechanisms. We show that, relative to the CD32a arginine (R)-131 (CD32aR) variant, CD32aH more avidly bound human (h) IgG1 IC and formed a ternary complex with the neonatal Fc receptor (FcRn) under acidic conditions. In primary human and mouse cells, both CD32a variants required FcRn to induce innate and adaptive immune responses to hIgG1 ICs, which were augmented in the setting of CD32aH. Conversely, FcRn induced responses to IgG IC independently of classical FcγR, but optimal responses required FcRn and FcγR. Finally, FcRn blockade decreased inflammation in a rheumatoid arthritis model without reducing circulating autoantibody levels, providing support for FcRn's direct role in IgG IC-associated inflammation. Thus, CD32a and FcRn coregulate IgG IC-mediated immunity in a manner favoring the CD32aH variant, providing a novel mechanism for its disease association.

Omeprazole inhibits IgE-mediated mast cell activation and allergic inflammation induced by ingested allergen in mice.

BACKGROUND: Patients with eosinophilic esophagitis have increased numbers of mucosal mast cells. Administration of the proton pump inhibitor omeprazole can reduce both esophageal mast cell and eosinophil numbers and attenuate type 2 inflammation in these subjects. OBJECTIVE: Given that maintenance of an acidic environment within granules is important for mast cell homeostasis, we sought to evaluate the effects of omeprazole on mast cell functions including development, IgE:FcεRI-mediated activation, and responses to food allergen. METHODS: Mast cell degranulation, cytokine secretion, and early signaling events in the FcεRI pathway, including protein kinase phosphorylation and Ca2+ flux, were measured after IgE crosslinking in murine bone marrow-derived mast cells and human cord blood-derived mast cells. The effects of omeprazole on these responses were investigated as was its impact on mast cell-dependent anaphylaxis and food allergy phenotypes in vivo. RESULTS: Murine and human mast cells treated with omeprazole exhibited diminished degranulation and release of cytokines and histamine in response to allergen. In murine mast cells, phosphorylation of protein kinases, ERK and SYK, was decreased. Differentiation of mast cells from bone marrow progenitors was also inhibited. IgE-mediated passive anaphylaxis was blunted in mice treated with omeprazole as was allergen-induced mast cell expansion and mast cell activation in the intestine in a model of food allergy. CONCLUSIONS: Our findings suggest that omeprazole targets pathways important for the differentiation and activation of murine mast cells and for the manifestations of food allergy and anaphylaxis.

Optimized Culture Conditions for Improved Growth and Functional Differentiation of Mouse and Human Colon Organoids.

Background & Aims: Diligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids. Methods: Mouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors. Results: In contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures. Conclusion: Our study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.

A Shocking Type of Communication.

In allergic patients, small amounts of allergen activate mast cells and trigger an immune cascade that can result in anaphylactic shock. In a recent issue of Science, Choi et al. (2018) show that dendritic cells sample the lumen of blood vessels and use microvesicles to trigger mast cell degranulation and anaphylaxis.

A Distinct Esophageal mRNA Pattern Identifies Eosinophilic Esophagitis Patients With Food Impactions.

Eosinophilic esophagitis (EoE), a Th2-type allergic immune disorder characterized by an eosinophil-rich esophageal immune infiltrate, is often associated with food impaction (FI) in pediatric patients but the molecular mechanisms underlying the development of this complication are not well understood. We aim to identify molecular pathways involved in the development of FI. Due to large variations in disease presentation, our analysis was further geared to find markers capable of distinguishing EoE patients that are prone to develop food impactions and thus expand an established medical algorithm for EoE by developing a secondary analysis that allows for the identification of patients with food impactions as a distinct patient population. To this end, mRNA patterns from esophageal biopsies of pediatric EoE patients presenting with and without food impactions were compared and machine learning techniques were employed to establish a diagnostic probability score to identify patients with food impactions (EoE+FI). Our analysis showed that EoE patients with food impaction were indistinguishable from other EoE patients based on their tissue eosinophil count, serum IgE levels, or the mRNA transcriptome-based p(EoE). Irrespectively, an additional analysis loop of the medical algorithm was able to separate EoE+FI patients and a composite FI-score was established that identified such patients with a sensitivity of 93% and a specificity of 100%. The esophageal mRNA pattern of EoE+FI patients was typified by lower expression levels of mast cell markers and Th2 associated transcripts, such as FCERIB, CPA3, CCL2, IL4, and IL5. Furthermore, lower expression levels of regulators of esophageal motility (NOS2 and HIF1A) were detected in EoE+FI. The EoE+FI -specific mRNA pattern indicates that impaired motility may be one underlying factor for the development of food impactions in pediatric patients. The availability of improved diagnostic tools such as a medical algorithm for EoE subpopulations will have a direct impact on clinical practice because such strategies can identify molecular inflammatory characteristics of individual EoE patients, which, in turn, will facilitate the development of individualized therapeutic approaches that target the relevant pathways affected in each patient.

Experimental Models for Studying Food Allergy.

Immunoglobulin E-mediated food allergy is rapidly developing into a global health problem. Publicly available therapeutic intervention strategies are currently restricted to allergen avoidance and emergency treatments. To gain a better understanding of the disease pathophysiology so that new therapies can be developed, major research efforts have been put into studying food allergy in mice. Animal models should reflect the human pathology as closely as possible to allow for a rapid translation of basic science observations to the bedside. In this regard, experimental models of food allergy provide significant challenges for research because of discrepancies between the presentation of disease in humans and mice. The goal of this review is to give a summary of commonly used murine disease models and to discuss how they relate to the human condition. We will focus on epicutaneous sensitization models, on mouse strains that sensitize spontaneously to food as seen in humans, and on models in humanized animals. In summary, expanding the research toolbox of experimental food allergy provides an important step toward closing gaps in our understanding of the derailing immune mechanism that underlies the human disease. The availability of additional experimental models will provide exciting opportunities to discover new intervention points for the treatment of food allergies. (Cell Mol Gastroenterol Hepatol 2018;x:x).

An algorithm for the classification of mRNA patterns in eosinophilic esophagitis: Integration of machine learning.

BACKGROUND: Diagnostic evaluation of eosinophilic esophagitis (EoE) remains difficult, particularly the assessment of the patient's allergic status. OBJECTIVE: This study sought to establish an automated medical algorithm to assist in the evaluation of EoE. METHODS: Machine learning techniques were used to establish a diagnostic probability score for EoE, p(EoE), based on esophageal mRNA transcript patterns from biopsies of patients with EoE, gastroesophageal reflux disease and controls. Dimensionality reduction in the training set established weighted factors, which were confirmed by immunohistochemistry. Following weighted factor analysis, p(EoE) was determined by random forest classification. Accuracy was tested in an external test set, and predictive power was assessed with equivocal patients. Esophageal IgE production was quantified with epsilon germ line (IGHE) transcripts and correlated with serum IgE and the Th2-type mRNA profile to establish an IGHE score for tissue allergy. RESULTS: In the primary analysis, a 3-class statistical model generated a p(EoE) score based on common characteristics of the inflammatory EoE profile. A p(EoE) ≥ 25 successfully identified EoE with high accuracy (sensitivity: 90.9%, specificity: 93.2%, area under the curve: 0.985) and improved diagnosis of equivocal cases by 84.6%. The p(EoE) changed in response to therapy. A secondary analysis loop in EoE patients defined an IGHE score of ≥37.5 for a patient subpopulation with increased esophageal allergic inflammation. CONCLUSIONS: The development of intelligent data analysis from a machine learning perspective provides exciting opportunities to improve diagnostic precision and improve patient care in EoE. The p(EoE) and the IGHE score are steps toward the development of decision trees to define EoE subpopulations and, consequently, will facilitate individualized therapy.

FOXP3+ Tregs require WASP to restrain Th2-mediated food allergy.

In addition to the infectious consequences of immunodeficiency, patients with Wiskott-Aldrich syndrome (WAS) often suffer from poorly understood exaggerated immune responses that result in autoimmunity and elevated levels of serum IgE. Here, we have shown that WAS patients and mice deficient in WAS protein (WASP) frequently develop IgE-mediated reactions to common food allergens. WASP-deficient animals displayed an adjuvant-free IgE-sensitization to chow antigens that was most pronounced for wheat and soy and occurred under specific pathogen-free as well as germ-free housing conditions. Conditional deletion of Was in FOXP3+ Tregs resulted in more severe Th2-type intestinal inflammation than that observed in mice with global WASP deficiency, indicating that allergic responses to food allergens are dependent upon loss of WASP expression in this immune compartment. While WASP-deficient Tregs efficiently contained Th1- and Th17-type effector differentiation in vivo, they failed to restrain Th2 effector responses that drive allergic intestinal inflammation. Loss of WASP was phenotypically associated with increased GATA3 expression in effector memory FOXP3+ Tregs, but not in naive-like FOXP3+ Tregs, an effect that occurred independently of increased IL-4 signaling. Our results reveal a Treg-specific role for WASP that is required for prevention of Th2 effector cell differentiation and allergic sensitization to dietary antigens.

Eosinophilic esophagitis: published evidences for disease subtypes, indications for patient subpopulations, and how to translate patient observations to murine experimental models.

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder of the esophagus and commonly classified as a Th2-type allergy. Major advances in our understanding of the EoE pathophysiology have recently been made, but clinicians struggle with highly unpredictable therapy responses indicative of phenotypic diversity within the patient population. Here, we summarize evidences for the existence of EoE subpopulations based on diverse inflammatory characteristics of the esophageal tissue in EoE. Additionally, clinical characteristics of EoE patients support the concept of disease subtypes. We conclude that clinical and experimental evidences indicate that EoE is an umbrella term for conditions that are unified by esophageal eosinophilia but that several disease subgroups with various inflammatory esophageal patterns and/or different clinical features exist. We further discuss strategies to study the pathophysiologic differences as observed in EoE patients in murine experimental EoE. Going forward, models of EoE that faithfully mimic EoE subentities as defined in humans will be essential because mechanistic studies on triggers which regulate the onset of diverse EoE subpopulations are not feasible in patients. Understanding how and why different EoE phenotypes develop will be a first and fundamental step to establish strategies that integrate individual variations of the EoE pathology into personalized therapy.

Allergic skin sensitization promotes eosinophilic esophagitis through the IL-33-basophil axis in mice.

BACKGROUND: Eosinophilic esophagitis (EoE) is an allergic inflammatory disorder characterized by accumulation of eosinophils in the esophagus. EoE often coexists with atopic dermatitis, a chronic inflammatory skin disease. The impaired skin barrier in patients with atopic dermatitis has been suggested as an entry point for allergic sensitization that triggers development of EoE. OBJECTIVE: We sought to define the mechanisms whereby epicutaneous sensitization through a disrupted skin barrier induces development of EoE. METHODS: To elicit experimental EoE, mice were epicutaneously sensitized with ovalbumin (OVA), followed by intranasal OVA challenge. Levels of esophageal mRNA for TH2 cytokines and the IL-33 receptor Il1rl1 (St2) were measured by using quantitative PCR. Esophageal eosinophil accumulation was assessed by using flow cytometry and hematoxylin and eosin staining. In vivo basophil depletion was achieved with diphtheria toxin treatment of Mcpt8DTR mice, and animals were repopulated with bone marrow basophils. mRNA analysis of esophageal biopsy specimens from patients with EoE was used to validate our findings in human subjects. RESULTS: Epicutaneous sensitization and intranasal challenge of wild-type mice resulted in accumulation of eosinophils and upregulation of TH2 cytokines and St2 in the esophagus. Disruption of the IL-33-ST2 axis or depletion of basophils reduced these features. Expression of ST2 on basophils was required to accumulate in the esophagus and transfer experimental EoE. Expression of IL1RL1/ST2 mRNA was increased in esophageal biopsy specimens from patients with EoE. Topical OVA application on unstripped skin induced experimental EoE in filaggrin-deficient flaky tail (ft/ft) mice but not in wild-type control or ft/ft.St2-/- mice. CONCLUSION: Epicutaneous allergic sensitization promotes EoE, and this is critically mediated through the IL-33-ST2-basophil axis.

Electrophysiological Studies into the Safety of the Anti-diarrheal Drug Clotrimazole during Oral Rehydration Therapy.

BACKGROUND AND AIMS: Morbidity and mortality from acute diarrheal disease remains high, particularly in developing countries and in cases of natural or man-made disasters. Previous work has shown that the small molecule clotrimazole inhibits intestinal Cl- secretion by blocking both cyclic nucleotide- and Ca(2+)-gated K(+) channels, implicating its use in the treatment of diarrhea of diverse etiologies. Clotrimazole, however, might also inhibit transporters that mediate the inwardly directed electrochemical potential for Na(+)-dependent solute absorption, which would undermine its clinical application. Here we test this possibility by examining the effects of clotrimazole on Na(+)-coupled glucose uptake. MATERIALS AND METHODS: Short-circuit currents (Isc) following administration of glucose and secretagogues were studied in clotrimazole-treated jejunal sections of mouse intestine mounted in Ussing chambers. RESULTS: Treatment of small intestinal tissue with clotrimazole inhibited the Cl- secretory currents that resulted from challenge with the cAMP-agonist vasoactive intestinal peptide (VIP) or Ca(2+)-agonist carbachol in a dose-dependent fashion. A dose of 30 µM was effective in significantly reducing the Isc response to VIP and carbachol by 50% and 72%, respectively. At this dose, uptake of glucose was only marginally affected (decreased by 14%, p = 0.37). There was no measurable effect on SGLT1-mediated sugar transport, as uptake of SGLT1-restricted 3-O-methyl glucose was equivalent between clotrimazole-treated and untreated tissue (98% vs. 100%, p = 0.90). CONCLUSION: Treatment of intestinal tissue with clotrimazole significantly reduced secretory responses caused by both cAMP- and Ca(2+)-dependent agonists as expected, but did not affect Na(+)-coupled glucose absorption. Clotrimazole could thus be used in conjunction with oral rehydration solution as a low-cost, auxiliary treatment of acute secretory diarrheas.

Functions of dendritic-cell-bound IgE in allergy.

Immunoglobulin E (IgE) functions as an Fc-receptor-bound antigen sensor for mast cells and basophils, the classical effector cells of allergy. A cell-bound IgE pool is formed when monomeric IgE binds to FcɛRI, the high affinity IgE Fc receptor on these cells, and minor amounts of antigen are sufficient to trigger the pro-allergic innate IgE effector axis. Additionally, FcɛRI is constitutively expressed on human dendritic cells (DCs), and thus the latter cell type also receives signals via cell-bound IgE. Notably, steady-state expression of FcɛRI on DCs is absent in SPF-housed mice. How DCs integrate IgE/FcɛRI-derived signals into their sentinel functions as gatekeepers of immunity was therefore only recently studied with transgenic mice that phenocopy human FcɛRI expression. In this review, we summarize advances in our understanding of the functions of DC-bound IgE which demonstrate that IgE-mediated activation of DCs in allergic Th2-type inflammation appears to be immune regulatory rather than pro-inflammatory.

Fatal autoimmunity in mice reconstituted with human hematopoietic stem cells encoding defective FOXP3.

Mice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific "humanized" mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. To address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHC II) and instead expresses human leukocyte antigen DR1 (HLA-DR1). These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T-cell reconstitution, delayed-type hypersensitivity responses, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, associated with aberrant FOXP3 function, mice developed a lethal inflammatory disorder with multiorgan involvement and autoantibody production mimicking the pathology seen in affected humans. This humanized mouse model permits in vivo evaluation of immune responses associated with genetically altered HSCs, including primary immunodeficiencies, and should facilitate the study of human immune pathobiology and the development of targeted therapeutics.

A single glycan on IgE is indispensable for initiation of anaphylaxis.

Immunoglobulin ε (IgE) antibodies are the primary mediators of allergic diseases, which affect more than 1 in 10 individuals worldwide. IgE specific for innocuous environmental antigens, or allergens, binds and sensitizes tissue-resident mast cells expressing the high-affinity IgE receptor, FcεRI. Subsequent allergen exposure cross-links mast cell-bound IgE, resulting in the release of inflammatory mediators and initiation of the allergic cascade. It is well established that precise glycosylation patterns exert profound effects on the biological activity of IgG. However, the contribution of glycosylation to IgE biology is less clear. Here, we demonstrate an absolute requirement for IgE glycosylation in allergic reactions. The obligatory glycan was mapped to a single N-linked oligomannose structure in the constant domain 3 (Cε3) of IgE, at asparagine-394 (N394) in human IgE and N384 in mouse. Genetic disruption of the site or enzymatic removal of the oligomannose glycan altered IgE secondary structure and abrogated IgE binding to FcεRI, rendering IgE incapable of eliciting mast cell degranulation, thereby preventing anaphylaxis. These results underscore an unappreciated and essential requirement of glycosylation in IgE biology.

Accuracy of digital mRNA profiling of oesophageal biopsies as a novel diagnostic approach to eosinophilic oesophagitis.

BACKGROUND: Quantification of tissue eosinophils remains the golden standard in diagnosing eosinophilic oesophagitis (EoE), but this approach suffers from poor specificity. It has been recognized that histopathological changes that occur in patients with EoE are associated with a disease-specific tissue transcriptome. OBJECTIVE: We hypothesized that digital mRNA profiling targeted at a set of EoE-specific and Th2 inflammatory genes in oesophageal biopsies could help differentiate patients with EoE from those with reflux oesophagitis (RE) or normal tissue histology (NH). METHODS: The mRNA expression levels of 79 target genes were defined in both proximal and distal biopsies of 196 patients with nCounter® (Nanostring) technology. According to clinicopathological diagnosis, these patients were grouped in a training set (35 EoE, 30 RE, 30 NH) for building of a three-class prediction model using the random forest method, and a blinded predictive set (n = 47) for model validation. RESULTS: A diagnostic model built on ten differentially expressed genes was able to differentiate with 100% sensitivity and specificity between conditions in the training set. In a blinded predictive set, this model was able to correctly predict EoE in 14 of 18 patients in distal (sensitivity 78%, 95% CI 52-93%) and 16 of 18 patients in proximal biopsies (sensitivity 89%, 95% CI 64-98%), without false-positive diagnosis of EoE in RE or NH patients (specificity 100%, 95% CI 85-100%). Sensitivity was increased to 94% (95% CI 71-100%) when either the best predictive distal or proximal biopsy was used. CONCLUSION AND CLINICAL RELEVANCE: We conclude that mRNA profiling of oesophageal tissue is an accurate diagnostic strategy in detecting EoE.